Journal: bioRxiv

Article Title: Targeting CD74-positive macrophages improves neoadjuvant therapy in cervical cancer as revealed by single-cell transcriptomics analysis

doi: 10.1101/2023.11.09.566505

Figure Lengend Snippet: (A) Venn diagram of the 18 candidate genes shared by the Macro 1, Strong, and Positive subgroups. (B) Violin plot of the CD74, GRN, CYBA, and CTSH expression in the Strong, Moderate, and Weak subgroups. (C) The normalized CD74 expression level after pre-treatment, NACT, and anti-PD-1 Ab combination therapy (NACT+anti-PD-1 Ab) in T2, T3, and T4 patients. The patient T3 samples received two rounds of NACT and anti-PD-1 Ab combination therapy were analyzed. (D) UMAP projection of the CD74 expression in macrophage subgroups. The expression plot was split based on the Strong, Moderate, and Weak subgroups. (E) The unsupervised transcriptional trajectory of macrophages from Monocle (version 3), colored by the pseudo-time. The circle labeled 1 indicated the start of the trajectory. (F) UMAP projection of the normalized expression of CD74, GRN, NPC2, CYBA, CTSH, and LAPTM5 in macrophage subgroups. The gray curve indicates the pseudo-time trajectory. (G) The CD74 and GRN expression dynamic change along with the pseudo-time, colored by the Strong, Moderate, and Weak subgroups. (H) The immunofluorescence assay showed the CD74 (red) and CD68 (green) expression in the tissues of patient T4 receiving NACT and anti-PD-1 Ab combination therapy. The green boxes represent CD74 negative macrophages, and the white boxes represent macrophages expressing CD74. (I) The CD74 mRNA level in HeLa or CaSki cells cocultured with macrophages derived from THP-1 cells. Anti-PD-1 Ab and CDDP were used to establish a model of NACT combined with immunotherapy in vitro. (J) The percentage of CD74-positive macrophages cocultured with CaSki cells was detected by flow cytometry. (K) The immunofluorescence assay showed the CD74 (red) and CD68 (green) expression of subcutaneous tumors treated with CDDP and/or anti-PD-1 Ab. (L) Flow cytometry revealed the number of CD74-positive macrophages in single-cell suspensions isolated from mice subcutaneous tumors. The p-value was obtained by a two-tailed unpaired Student’s t-test, and the results are presented as the mean ±SD.

Article Snippet: Anti-mouse PD-1 (CD279)-InVivo (10 mg/kg, every other day, Selleck, China) and Cisplatin (7 mg/kg, every four days, MedChemExpress, China) were injected into the abdominal cavity of mice to build a NACT combined with PD-1 therapy model. Milatuzumab (anti-CD74) (15 mg/kg, every other day, MedChemExpress, China) was used to block the expression of CD74.

Techniques: Expressing, Labeling, Immunofluorescence, Derivative Assay, In Vitro, Flow Cytometry, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting CD74-positive macrophages improves neoadjuvant therapy in cervical cancer as revealed by single-cell transcriptomics analysis

doi: 10.1101/2023.11.09.566505

Figure Lengend Snippet: (A, B) Phagocytosis ability detected by flow cytometry. Macrophages derived from THP-1 cells were co-cultured with SiHa cells knockdown (A) or overexpressing (B) CD74. Phagocytosis efficiency was quantified by the percentage of double fluorophore-positive macrophages in the upper right quadrant. (C) The phagocytic function of macrophages co-cultured with SiHa cells treated with anti-CD74 was measured by flow cytometry. (D) Density plot of the M1 and M2 ssGSEA score in the Strong, Moderate, and Weak subgroups. (E) M1/M2 ssGSEA ratio of the macrophages in the pretreatment, NACT, and anti-PD-1 Ab combination group. (F, G) UMAP projection of the normalized density of CD74 expression (F), M1 score, and M2 score (G) in the macrophage subgroups. (H, I) CD86 (H) and CD206 (I) expression of THP-1-derived macrophages after CD74 overexpression. CD86 is the surface marker of M1 macrophages, while CD206 is the surface marker of M2 macrophages. (J, K) CD86 (J) and CD206 (K) expression of THP-1-derived macrophages after CD74 knockdown. (L) Phagocytosis efficiency of THP-1-derived macrophages cocultured with SiHa cells. CDDP-induced phagocytic function loss of macrophages was rescued by knocking down CD74. (M) Flow cytometry detected the differences in phagocytic function among the control, the CDDP, and the anti-CD74 Ab combined with the CDDP group. THP-1-derived macrophages were cocultured with SiHa cells. (N) The representative image of the subcutaneous tumor model. Mouse cervical cancer cells TC-1 were subcutaneously inoculated into immunocompetent CD74 humanized BALB/c-hCD74 mice. Each group consisted of 6 mice receiving CDDP or CDDP combined with intraperitoneal injected anti-CD74 Ab. Tumors of four mice in the drug-combination group became nonpalpable on Day 4 after administration. (O) Growth curve of subcutaneous tumors in mice. (P) Phagocytosis assay was performed using macrophages isolated from the mice’s spleen and mouse cervical cancer cell line TC-1. Phagocytosis efficiency was quantified by the percentage of Dil and Dio double fluorophore-positive THP-1-derived macrophages. The p-value was obtained by a two-tailed unpaired Student’s t-test, and the results are presented as the mean ±SD. *p<0.05, **p < 0.01, ***p<0.001

Article Snippet: Anti-mouse PD-1 (CD279)-InVivo (10 mg/kg, every other day, Selleck, China) and Cisplatin (7 mg/kg, every four days, MedChemExpress, China) were injected into the abdominal cavity of mice to build a NACT combined with PD-1 therapy model. Milatuzumab (anti-CD74) (15 mg/kg, every other day, MedChemExpress, China) was used to block the expression of CD74.

Techniques: Flow Cytometry, Derivative Assay, Cell Culture, Knockdown, Expressing, Over Expression, Marker, Control, Injection, Phagocytosis Assay, Isolation, Two Tailed Test

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 3. Comparison of HEK cell responses expressing wild-type and mutagenized form of Orco. (a)— Universal Orco agonist, VUAA1, elicits dose-dependent Ca++ i increase in HEK293A cells expressing either CpomOrco (blue) or CpomOrcoQ417H (red). (b)—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation for CpomOrco (blue smooth line) or CpomOrcoQ417H (red smooth line) respectively, providing maximum responses of ~ 6.3 ± 0.1 and ~ 3.9 ± 1.3 ∆F, EC50s of ~ 157.1 ± 3.58 and ~ 261.8 ± 165.6 µM and Hill coefficients of ~ 2.4 ± 0.1 and ~ 2.1 ± 1.9; total number of cells analysed: N = 123 and 94. (c)—Effects of Pear ester on the activity of CpomOrco+OR3 (blue) and CpomOrcoQ417H+OR3 (red) heteromers. (d)—Pear ester concentration dependencies. Data points represent the mean response amplitudes (± SE) of cells from at least three experiments. Data were fit to a Hill equation providing the following parameters: maximum responses of ~ 6.6 ± 0.3 and 3.7 ± 0.1 ∆F; EC50s of ~ 210 ± 14.2 and ~ 733.5 ± 26.9 µM; Hill coefficients of ~ 4.6 ± 2.4 and ~ 4.6 ± 0.4, for CpomOR3/CpomOrco (blue smooth line) or CpomOR3/CpomOrcoQ417H (red smooth line) respectively. Total number of cells analysed: N = 160 and 262. Traces in A and C represent the mean responses of cells from one experiment. Data in B and D were not normalized. Scales in B and D are different.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Comparison, Expressing, Concentration Assay, Activity Assay

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 4. Testing pH sensitivity of HEK cell expressing wild-type and mutagenized form of Orco. (a)— Decrease in fluorescence intensity of the pH sensitive probe, BCECF, possibly reflects acidification of cytoplasm in response to low pH extracellular conditions. (b)—Comparison of VUAA1 concentration dependencies obtained after 30 min incubation at low pHe for CpomOrco or CpomOrcoQ417H. Left panels—VUAA1 activated calcium responses. Right panels—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation with the following parameters: maximum responses of ~ 1.3 ± 0.7 and ~ 1.8 ± 0.2 ∆F; EC50s of ~ 260 ± 187 and ~ 263.3 ± 45.3 µM; Hill coefficients of ~ 2.5 ± 4.3 and ~ 2.2 ± 0.5, for CpomOrco (blue smooth line, n = 84) or CpomOrcoQ417H (red smooth line, n = 63) respectively. Constraints were applied to fit greatly scattered data in B. Traces in B (left panels) represent the mean responses of cells from one experiment. Data in B (right panels) were not normalized. Concentration dependencies obtained in physiologically relevant control conditions were taken from Fig. 3.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Expressing, Fluorescence, Comparison, Concentration Assay, Incubation, Control